1. Field of Invention
Leukocyte adhesion deficiency (LAD), formerly known as bovine granulocytopathy syndrome, was identified in cattle, and was found to cause unthriftiness and death at an early age [Hagemoser, W. A. et al., "Granulocytopathy in a Holstein heifer." J. Am. Vet. Med. Assoc., vol. 183, pp. 1093-1094 (1983); Nagahata, H. et al., "Bovine granulocytopathy syndrome: Neutrophil dysfunction in Holstein Friesian calves." J. Vet. Med. A, vol. 34, pp. 445-451 (1987); Takahashi, K. et al., "Bovine granuloctypathy syndrome of Holstein-Friesian calves and heifers." Jpn. J. Vet. Sci., vol. 49, pp. 733-736 (1987)]. Recently, this disease was shown to be equivalent to human LAD disease [Kehrli, M. E., Jr. et al., "Molecular definition of the bovine granulocytopathy syndrome: Identification of deficiency of the Mac-1 (CD11b/CD18) glycoprotein." Am. J. Vet. Res., vol. 51, pp. 1826-1836 (1990)]. In humans, LAD is an autosomal recessive genetic disease which results from various distinct mutations in the gene for the leukocyte adhesion molecule, CD18 [Anderson, D. C. et al., "The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features." J. Infect. Dis., vol. 152, pp. 668-689 (1985)]. Preliminary studies indicated that there is only one defective allele in cattle and that the carrier frequency for LAD is high among Holstein cattle.
There are approximately 1100 young bulls introduced per year into progeny milk production testing programs in the United States. To achieve this number of bulls, approximately 1500 bulls per year are evaluated by bull stud organizations as candidates for the young sire evaluation program. To eliminate bovine leukocyte adhesion deficiency (BLAD) from the Holstein population, it is essential to know the BLAD carrier status of these bulls and also of a significant number of cows. Up to 20% of the Holstein Friesian cattle in the world may be carriers of this undesirable trait.
This invention relates to identifying a point mutation in the CD18 gene responsible for causing bovine leukocyte adhesion deficiency (BLAD) and to procedures used for testing and identifying cattle alleles attributable to BLAD.
2. Description of the Prior Art
Prior to this invention, the only test for detecting carriers utilized flow cytometry. Although this technique works well for detecting cattle with BLAD, its ability to distinguish BLAD carriers from normal cattle is limited because such testing is cumbersome and results are often ambiguous.
Recent developments in molecular biology have made it possible to rapidly and conclusively screen individuals for particular genetic defects. To make use of this technology, the genetic defect must be defined at the DNA level. Once this has been accomplished, various techniques can be employed to screen animals for the characteristic DNA sequence and thus their genetic status, i.e., homozygous normal, heterozygous carrier, or homozygous diseased, will be known.
Bovine citrullinemia, another bovine autosomal recessive disease, was recently defined at the DNA level [Dennis, J. A. et al., "Molecular definition of bovine argininosuccinate synthetase deficiency." Proc. Natl. Acad. Sci. U.S.A., vol. 86, pp. 7947-7951 (1989)]. A point mutation in the gene for bovine argininosuccinate synthetase was found to be the basis for this disease. By knowing the site of this mutation and adjacent DNA sequence information, a diagnostic test was developed. This test utilized DNA amplification followed by restriction enzyme digestion, and made identification of normal, carrier, and citrullinemic cattle possible. Citrullinemia is a separate genetic defect that is not linked to BLAD.